Staphylococcus aureus and Staphylococcus epidermidis are opportunistic bacterial pathogens responsible for a wide array of human and animal diseases. Importantly, S. aureus and S. epidermidis are the two leading causes of hospital-associated infections, which significantly increase morbidity and treatment costs. Exopolysaccharides are important mediators of staphylococcal infections; specifically, polysaccharide intercellular adhesin (PIA) enhances biofilm formation and immune evasion, while capsule facilitates immune evasion, colonization, and persistence. Staphylococcal PIA is synthesized when tricarboxylic acid (TCA) cycle activity is repressed. In contrast, synthesis of the S. aureus capsular polysaccharide requires TCA cycle activity. Interestingly, PIA and capsule are derived from the same amino sugar (i.e., UDP-N-acetyl-glucosamine. Synthesis of both exopolysaccharides is modulated by the availability of nutrients, oxygen, and iron. Similarly, TCA cycle activity is regulated by the availability of nutrients, oxygen, and iron and by certain stress-inducing stimuli such as heat, ethanol, and antibiotics. The linkage of TCA cycle activity and exopolysaccharide synthesis and the susceptibility of the TCA cycle to environmental inactivation lead us to hypothesize that one mechanism by which staphylococci perceive external environmental change is through alterations in TCA cycle activity. These changes in TCA cycle activity alter the bacterial metabolome increasing or decreasing the intracellular concentrations of metabolites and co- factors that are sensed by regulatory proteins, which increase or decrease exopolysaccharide biosynthesis. The aims of this proposal are to identify the TCA cycle metabolites that control exopolysaccharide synthesis and determine which regulatory proteins are responding to these TCA cycle metabolites to regulate exopolysaccharide synthesis. To achieve these aims, we will use an integrated approach combining systems biology, biochemistry, bioinformatics, and genetics. The rationale for the proposed research is to fill the gap in our understanding of how environmental conditions affect the bacterial metabolic status and, in turn, how the metabolic status affects staphylococcal exopolysaccharide biosynthesis. Understanding this will aid in our long-term goal, which is to design therapeutic strategies targeting staphylococcal metabolism and metabolic responsive regulators that will facilitate bacterial killing by antibiotics and the host immune system.